title: Luminal Ca2+ protects against thapsigargin inhibition in neuronal endoplasmic reticulum.
abstract: Thapsigargin is a specific and potent inhibitor of sarco/endoplasmic reticulum Ca2+-ATPases. However, in whole rat brain microsomes, 1 microM thapsigargin had no significant effect on the 10-min time course of ATP-dependent Ca2+ uptake in the absence of the luminal Ca2+ chelator oxalate. In contrast, 50 mM oxalate resolved a thapsigargin-sensitive Ca2+ uptake rate (IC50 approximately 1 nM thapsigargin) five times that of a thapsigargin-insensitive rate. This remaining approximately 20% of the total ATP-dependent accumulation was insensitive to thapsigargin (up to 10 microM), slightly less sensitive to vanadate inhibition, and unresponsive to 5 microM inositol 1,4,5-trisphosphate or 10 mM caffeine. Measuring both 12-min Ca2+ uptake and initial Ca2+ uptake rates, the apparent thapsigargin sensitivity increased as oxalate concentrations increased from 10 to 50 mM, corresponding to a range of luminal free Ca2+ concentrations of approximately 300 down to 60 nM. Addition of oxalate during steady-state 45Ca accumulation rapidly resolved the aforementioned thapsigargin sensitivity. These results strongly suggest that luminal Ca2+ may protect a large portion of neuronal endoplasmic reticulum Ca2+ pumps against thapsigargin inhibition. Although high [Ca2+] has been previously shown to protect against thapsigargin inhibition in several reticular membrane preparations, our results suggest that luminal Ca2+ alone is responsible for mediating this effect in neurons.
authors.affiliation: Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Wells
authors.forename: R F
authors.initials: RF
authors.lastname: Abercrombie
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001921:Brain / Q000166:cytology / Q000378:metabolism; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002132:Calcium Radioisotopes; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism / Q000494:pharmacology; D002451:Cell Compartmentation; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D009474:Neurons / Q000378:metabolism*; D010070:Oxalates / Q000494:pharmacology; D051381:Rats; D019284:Thapsigargin / Q000494:pharmacology*; D014638:Vanadates / Q000494:pharmacology
chemical_list: D002132:Calcium Radioisotopes; D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D010070:Oxalates; D002110:Caffeine; D014638:Vanadates; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium